Development and Genetic Changes of Isolated Aberrant Crypts Based on Clonal Analysis

1. 大鼠正常结肠隐窝和dmh诱导的ACF的发育：研究表明，正常出生后大鼠结肠隐窝通过裂变机制自我繁殖，dmh诱导的ACF也是如此。PhlP诱导的大鼠ACF分布：在给药后12周和25周，PhlP诱导的大鼠ACF主要分布在远端结肠，在给药后50周，主要分布在近端结肠。随着时间的推移，远端结肠ACF数量减少，近端结肠ACF数量增加。50周时，肿瘤大多局限于近端结肠。提示早期远端ACF是可逆的，部分晚期ACF可能发展为肿瘤。嵌合小鼠结肠隐窝的分离及c3h特异性单克隆抗体（CSA）克隆分析：嵌合小鼠的正常单结肠隐窝为克隆，粘膜呈两组来自同一克隆来源的隐窝拼凑而成。根据我们的研究，在dmh诱导的嵌合小鼠ACF中，所有隐窝都是由一个菌株的八个起源构成的。利用微卫星多态性分析嵌合小鼠结肠隐窝克隆：利用PCR反应，发现菌株间多态的DNA微卫星标记D8Mit4可区分嵌合小鼠离体隐窝克隆来源。结果与CSA染色结果一致。结论：基于以上结果，我们推测ACF是由单个结肠隐窝通过裂变机制单克隆发育而来。CSA染色结果证实了这一假设。从ACF的分布、形态、遗传变化和克隆性等方面研究ACF作为结肠肿瘤癌前病变的可逆性和不可逆性。

1. Development of normal colonic crypts and DMH-induced ACF in rats : it was shown that normal postnatal rat colonic crypts reproduced themselves by fission mechanism and as was also the case with DMH-induced ACF.2. Distribution of PhlP-induced rat ACF : Intragastrical administration of PhlP induced rat ACF mainly in the distal colon at weeks 12 and 25 and predominanly in the proximal colon at week 50 after the treatment. The number of ACF decreased in the distal colon and increased in the proximal colon with time. Cancers were mostly localized in the proximal colon at week 50. It suggested that the early distal ACF were reversible and some of late distal ACF might have developed into cancers.3. Colonic crypts isolation and clonal analysis with C3H-specific monoclonal antibody (CSA) in chimeric mice : Normal single colonic crypt of chimeric mice were revealed as clonal and the mucosa showed like a patchwork consisting of two groups of crypts derived from the same clonal origin. As far as we investigated, all crypts were constituted with eighter orgin of a strain in a DMH-induced ACF in chimeric mice.4. Clonal analysis of colonic crypts in chimeric mice by microsatellite polymorphism : Using PCR reaction, a DNA microsatellite marker D8Mit4, polymorphic among strains, could distinguish the clonal origin of isolated crypts of chimeric mice. The result was identical with that judged by CSA staining.5. Conclusion : With the above results, we hypothesized that ACF were monoclonally developed from a single colonic crypt by fission mechanism. The results of CSA staining proved this hypothesis to be true. Investigation of ACF in terms of distribution, morphology, genetic change, and clonality may reveal the character of reverlibility and irreversiblity of ACF as a precancerous lesion of colonic tumor.